Assessment of Anti-Coccidial Efficacy of Ethanolic Extract of <i>Aloe</i> <i>vera</i> Leaf in Kabir Chicken in Cameroon

In developing countries, poultry production is largely based on traditional extensive poultry production systems (Bawankar et al., 2014). Village chicken represent an appropriate system to provide high-quality protein to the fast growing human population. They also provide income to resource-poor smallholder farmers, especially women. However, high mortality rates, mainly due to diseases, including coccidiosis, constitute one of the greatest constraints on chicken development. Coccidiosis causes considerable economic loss in the poultry industry, especially in broiler chicken as it is associated with reduced growth rate and impaired feed conversion thus leading to poor performance of chicken and mortality (Usman et al., 2011). Chicken are susceptible to at least nine species of coccidia. The commonest species are Eimeria tenella, which causes caecal coccidiosis and E. acervulina as well as E. maxima which cause chronic intestinal coccidiosis. Currently, coccidiosis control programmes largely rely on chemotherapy and immunoprophylaxis. However, the development of drug resistance in strains in the field and the withdrawal period for these drugs prior to slaughter necessitate the exploration of alternative methods of treatment such as the use of medicinal plant extracts for controlling the disease. Usman et al. (2011) tested fifteen herbal extracts for anticoccidial activity in chicks and had varying degrees of results. Plant extracts such as Carica papaya, Moringa indica, Mentha arvensis and Aloe vera have been used traditionally to treat various gastrointestinal ailments in man and animals in different parts of the world (Hashemabadi and Kaviani, 2008).

Aloe vera is a cactus-like plant. It is stemless with triangular, fleshy leaves ranging in colour from grey-green to bright green and the margins of the leaves have small white ‘teeth’. The leaves are composed of three layers: an inner gel, a yellow sap and the outer thick layer of 15-20 cells known as rind (Surjushe et al., 2008; Hashemabadit and Kaviani, 2010).

Reports on the medicinal properties of A. vera include the external healing of different kinds of wounds, excellent soothing of minor burns, scrapes, ulcers, and alleviation of arthritis, constipation and piles (Hashemabadit and Kaviani, 2010). The leaf and juice of A. vera have been used in animals internally or externally. It is believed to possess pharmacological antibacterial, antivenin, and immunological properties. Some resource-poor smallholder farmers usually use Aloe species to reduce chicken mortalities. Common examples of Aloe used in Southern Africa include A. vera excelsa, Aloe christiana, and Aloe spicata which are easy to use and are available all year round (Trivedi et al., 2008). However, the potential for using A. vera in livestock health management is not widely documented (Bawankar et al., 2014). There is therefore a need to investigate and document the therapeutic importance of the aloes, which may in turn result in the preservation and protection of the natural plant resource-base. Smallholder farmers are able to contribute in the conservation efforts if they use the herb for ethno-veterinary and medicinal purposes. Although ethno-veterinary medicines are widely used, the optimum dosage and the dose response characteristics are not known (Gueye and Van’t-Hooft, 2000; Hashemabadit and Kaviani, 2010).

Commonly, haematological parameter measurements provides valuable information in human and animal medicine. Unfortunately, due to lack of information of such parameters, blood profile is not used widely in avian medicine (Mukherjee and Wahile, 2006; Kayode et al., 2009). Although there is limited information in this field, evaluation of blood profiles of broiler strains has been assessed by some studies. Significant increases in haematological parameters with age have been reported in broiler chicks such as WBC counts including lymphocytes, monocytes, heterophils, eosinophils and basophils (Arczewska-Wlose and Swiatkiewz, 2012; Ajay et al., 2013) and RBC, haemoglobin (Hb) and packed cell volume (PCV) as reported by Aliasghar et al. (2012). Such studies need to be validated in different localities in order to appreciate the medicinal value of the plant extract as this could reduce the challenges faced by chicken and chicken farmers as a result of coccidiosis. Against this background, this study was undertaken to evaluate the anticoccidial efficacy of the ethanolic extract of A. vera in Kabir chicken by assessing the oocyst load, growth and haematological parameters.

The study was carried out in the Africa Brazil Marketplace project poultry farm located in Lyongo village, Buea, South West Region, Cameroon. The locality is located at the foot of Mount Cameroon and is very rich in vegetation. The experimental animals consisted of Cameroonian Kabir chicken aged 9 weeks comprising 8 fowls per experimental group and of both sexes. They were of mixed breed, displaying a variety of feathering and plumage colours.

After leaving the hatchery the experimental chicks were grown under uniform brooder conditions from a day old to experimental ages. The birds were housed in a disinfected deep litter system with wood shavings as bedding material. Each treatment occupied an area of 2.25 m2 where feed and water were provided ad libitum. Incandescent bulbs were used to provide light and heat day and night during the brooding period.

Kabir chicken aged 9 weeks old and of both sexes with naked necks were divided into 6 pens (Groups) containing 8 chicken each. Groups 1-5 (T1-T5) were infected with approximately 3200 Eimeria oocysts at the 8th week. The faeces of the chicken was monitored on a daily basis for the presence of oocysts. On the 4th day post-infection, oocysts were found in the stool of the birds and treatment commenced and went on for six days after which blood was extracted and the chicken sacrificed for carcass analyses. Groups 1-3 (T1-T3) were given the ethanolic plant extract of A. vera in different doses of 0.16 g, 0.32 g and 0.84 g/chicken/day respectively. Group 4 (T4) was given the commercial anticoccidial drug (Sulfaquinoxalina-LH); 1 tea spoonful of powder in 2.5 mL of water in their drinkers for four days according to the manufacturer’s instruction. Group 5 (T5) was the control group which was infected with the Eimeria parasites but not treated at all and group 6 (T6), the neutral group was not infected at all, but given only water. All the chicken in each group were of equal numbers (8/group), sexes and plumage, same feed intake and composition, under the same environmental conditions with ad libitum access to feed and water. A reservoir chicken for coccidian oocysts was used and oocyst output per gram of faeces was quantified using the McMaster technique and found to supply mean output of 3200 opg of faeces. This reservoir chicken was treated for other parasitic, bacterial and viral infections except that of coccidia. A gram of filtered faeces was obtained from the reservoir chicken, dissolved and administered orally to each chicken in T1-T5 except T6. By implication, the chicken received approximately 3200 Eimeria oocysts. The McMaster technique was used to monitor the oocyst output in the stool of the infected chicken (Christensen et al., 1987).

Fresh A. vera leaves were harvested, dried in gentle heat, ground and sieved. 95% of ethanol was diluted to 70% ethanol using Gay-Lussac’s dilution method to form alcoholic water which was used for the preparation of the ethanolic extract (http://www.spc.ac-aix-marseille.fr/labospc/spip.php?article329 accessed May, 2015). A. vera leaf powder (500 g) was put into 1000 ml of 70% ethanol and covered to avoid evaporation. This was macerated and stirred after every 6 hours, for 72 hours after which, the mixture was filtered through filter papers and the liquid was evaporated to dryness using an evaporator at 40oC. The powder was given in 0.16 g, 0.32 g and 0.84 g for T1, T2 and T3 respectively per chicken per day. T4 contained sulfaquinoxalina-LH and was administered 1 teaspoon full daily in 2.5 L of water. It was taken for 4 days. The weights of these birds were taken after every two days using a sensitive balance while oocysts per gram of faeces was quantified by the McMaster counting technique (Christensen et al., 1987).

At the end of the experiment, chicken from each treatment were sacrificed, blood samples were collected from their aortic veins into EDTA tubes for haematological analysis. The chicken were de-feathered, and opened for the evaluation of carcass and internal organ parameters to evaluate the effects of these parasites and different A. vera extract doses on their weights. The internal organs were carefully removed, observed and weighed using an electronic balance (Ohaus CS200, USA). The organs concerned were live weight (LWT), carcass weight (CWT), heart (HT), liver (LV), lungs (LGS), spleen (SPL), fabricius bursa (FB), gizzard (GZ), proventriculus (PV), thymus (TH), caecal weight (CEWT), caecal length (CEL), kidney (KN), empty caecal weight (ECEWT) and bile (BL). The blood was transported in ice and carried to the Laboratory of Animal Health (LASAN) of the Department of Animal Production, of the Faculty of Agronomy and Agricultural Sciences, University of Dschang, Cameroon for analysis. Haematological parameters (WBC, RBC and Hb) were analysed using the haematology analyser machine for animals (Beckman Coulter, Coulter AC. T different Haematology Analyser, BR-13692A).

The data collected was entered and analysed using SPSS Package version 20. The data was subjected to ANOVA at 95% confidence level (P < 0.05) to compare means, and when there were significant differences between means, these were separated using the Duncan multiple range test. The mean oocyst counts that were calculated before and after the administration of the different extracts were used to evaluate percentage in reduction or reduction rate, using the formula below:

Reduction rate = (Initial oocyst counts-final oocyst counts x 100)/ (Initial oocyts counts)

Also, some production parameters such as the average feed intake (FI) which is the total amount of feed consumed by the chicken from the day of the administration of the plant extracts till the last day, average daily weight gain (AWG) between treatments were calculated. These parameters were calculated as follows:

Average daily weight gain (AWG) = Total weight gain per chicken per group/ Total number of days

Feed consumption rate (FCR) = FI / Total AWG

Where, FI = feed intake and AWG is average weight gain (Unigwe et al., 2014).

Table 1: The feed intake, cumulative average weight gain, growth rate and feed conversion ratio of the Kabir chickens treated with ethanolic A. vera leaf extract

Values in a column followed by the same superscripts are not significantly different (P > 0.05); SEM = standard error of means; FI = feed intake; AWG = average weight gain; AGR = average growth rate; FCR = feed conversion rate.

The Growth parameters of Eimeria-infected Kabir chicken treated with ethanolic A. vera leaf extract are presented in Table 1. Results obtained showed that there were significant differences in the feed intake between the different groups receiving the ethanolic A. vera leaf extract (P < 0.05). A significant difference (P = 0.014) was observed in the average weight gain between groups and these are shown by superscripts from Turkey multiple comparison test in Table 1. The highest average growth rate was observed in chicken of T3 (11.7%) while the lowest was observed in fowls of T5 (3.1%). Moreover, the highest feed conversion ratio was observed in T4 (18.6%) while the least was observed in fowls of T1 (7.1%).

The Carcass characteristics of Eimeria-infected Kabir chicken treated with ethanolic A. vera leaf extract are presented in Table 2. The results obtained revealed no significant difference in the weights of organs between groups at P > 0.05.

Table 2: Average weight of some internal organs of Eimeria- infected Kabir chicken treated with ethanolic extract of Aloe vera

Table 3: Reduction rate of Eimeria oocyst counts in Kabir chicken treated with ethanolic extracts of A. vera leaves

a, b on the same column, the values assigned the same letter are not significantly different (P > 0.05); H-L = high to low, M-H = Medium to high OPG

The final faecal oocyst counts of Eimeria-infected Kabir chicken treated with ethanolic A. vera leaf extract in different groups showed significant reduction in oocyst count between treatment groups (Table 3). The oocyst counts in the untreated group (T5) increased continuously from the initial count on Day 4 as opposed to the treated group. The highest oocyst count reduction rate was observed in T4 (98.7%) which received the standard anti-coccidial drug. Among the groups that received the plant extract, the highest oocyst reduction rate was observed in T3 (97.9%), and reduced in a dose-dependent manner to 97.8% in T2 and then 80.2% in T1, the differences in oocyst reduction between the treated groups (T1-T4) were not significant at p > 0.05 except with the untreated group at P < 0.05.

The haematological parameters for Eimeria-infected Kabir chicken treated with ethanolic extracts of A. vera leaves are presented in Table 4. There was a statistically significant difference in RBC and WBC counts at P ≤ 0.043 and P ≤ 0.031 respectively between experimental groups treated with ethanolic A. vera leaf extracts.

Coccidiosis constitutes a major health problem to the poultry industry and has primarily been controlled by the use of standard medication under field conditions in spite of limitations like drug resistance and other concerns in relation to food chain contamination. As a substitute, the use of plants and their products as immunomodulators and therapeutics have traditionally been used. According to Farnsworth’s report in 1999, more than 64% of the world’s population use botanical drugs to combat health problems. In this regard, therapeutic properties of A. vera have been studied in different animal models and human beings. These include anti-inflammatory immunomodulatory, wound healing, promotion of radiation damage repair, antibacterial, antiviral and antifungal (Arczewska-Włosek and Świątkiewicz, 2012).

Table 4: Effects of treatment with ethanolic A. vera leaf extracts on the RBC, WBC and Hb of Eimeria- infected Kabir chicken

The results of this study showed that the feed intake of the chicken between groups was significantly affected by the plant extracts. It can therefore be assumed that the beneficial effect (high percentage weight gain of 11. 7% in T3) of the A. vera extract obtained in infected chicken was as a consequence of its stimulating influence on the chickens’ appetite, but not necessarily as a result of its anticoccidial properties (Arczewska-Włosek and Świątkiewicz, 2012). In line with this finding, Guo et al. (2004), Alfifi (2007) and Durrani et al. (2007) reported significant differences in feed consumption rate of broilers fed with A. vera leaf and other herbal extracts.

Chicken of T3 that received the highest dose of the ethanolic A. vera leaf extract on the other hand, had the highest average percentage weight gain (11.7%) while T5 had the lowest with 3.1%. This could be attributed to the fact that the A. vera leaf extract has a feed stimulatory enzyme which assists in feed consumption rate and intake. As a result of significant food intake with A. vera this also led to a statistically significant average weight gain (P = 0.014) of the Kabir chicken. On the contrary, Sarad et al. (2008) in Tanzania reported no significant difference in food and water intake with fowls treated with A. vera extract. Chandrakesan et al. (2009) reported that coccidial challenge significantly affected body mass gain and feed conversion ratio of chicks. However, Kurkure et al. (2006) on the contrary reported that coccidial challenge had no effect on body mass gain and feed conversion on fowls treated with A. vera extract.

When carcass parameters were evaluated, the plant extracts did not have any significant effect on the major body organs although higher values were recorded in treated groups in a dose-dependent manner. Findings of the present study are in agreement with the reports of Sungirai et al. (2013) who all reported no significant differences in major body organs with broilers infected with oocysts and treated with Aloe extract.

Significantly (P < 0.05) higher body weight gain, dressed weight and lower feed conversion ratio were observed for chicken in the test group when compared with control groups. Similar findings have been reported by Wheeler et al. (1994), Guo et al. (2004), Chand et al. (2005), Jiang et al. (2005), Mehmet et al. (2005), Durrani et al. (2007) and Sarad et al. (2008). The higher body weight gain of the chicken given the ethanolic extract of A. vera could be due to better performance of the chicken and the diversified antimicrobial activities of Aloe gel. This was also demonstrated by Swaim et al. (1992) in chicken. A. vera juice is known for healing wounds caused by trophozoites at the level of the caeca which in this present work showed no significant difference in terms of empty caecal weights (ECEWT).

There was a significant reduction in faecal oocyst count with an increase in A. vera concentration. The reduction in oocyst count was probably as a result of the anti-coccidial activities of the A. vera extract. Sungirai et al. (2013) reported similar findings in Zimbabwe with treated and untreated groups of broiler chicken with Aloe extract. Gadzirayi et al. (2010) reported similar results after adding A. vera powder to the drinkers of experimental chicken. The same author also found out that A. excelsa was as good as a synthetic coccidiostat in controlling coccidiosis. It has been reported that Aloe juice contains organic chemicals known as 1, 8 dihydroxyanthraquinone and its derivatives include Aloe emodin, aloetic acid and isobarbaloin (Hamman, 2008; Yim et al., 2011). These chemical constituents in Aloe extract act as laxative agents by interacting with the gastrointestinal mucosa and inducing bowel motility. This leads to the quick discharge of coccidial oocysts that are lodged in faecal matter thereby reducing the oocyst count. Gadzirayi et al. (2010) reported similar findings with Aloe excelsa extract.

Results obtained also showed that ethanolic A. vera leaf extract had significant rise on the RBC (P = 0.048) and WBC (P = 0.024) counts of the chicken between treatments. In line with this study, Sarad et al. (2008) reported significantly higher WBC level in a similar study with experimental broilers treated with Aloe extract as compared to others. Sham et al. (2003), Gautam et al. (2004), Balwinder et al. (2005), Valle et al. (2005) and Durrani et al. (2007) also reported increased levels of immunities in chicken given various herbal extracts in drinking water. This is probably due to the fact that A. vera has immune stimulatory properties and it is this property that it uses to raise antibodies against coccidial infections. Since antibodies are raised to fight these parasites, it is therefore suggested that this is the reason for the high WBC in the Aloe treated group. Blood loss due to coocidial infection was also minimized and this is probably the reason for the higher RBC levels with respect to other groups.

Aloe vera extract proved to be of significant importance in fighting coccidiosis and showed a significant reduction of oocyst counts of 97.9% and significant increases in WBC and RBC levels. Thus, the extract could be used as organic alternative to synthetic chemicals to combat coccidiosis and improve chicken health especially in poor communities.

This work was supported by funds from the EMBRAPA–Brazil, provided for the execution of the ID 207 Africa Brazil Agricultural Innovation Marketplace project. The authors are also grateful to Dr. Ngoula Ferdinand and Mr. Thomas Tebug of the Laboratory of Animal Health (LASAN) of the Department of Animal Production, of the Faculty of Agronomy and Agricultural Sciences, University of Dschang, Cameroon where the haematological analysis was done. We are also grateful to Mr Fairo Dzekashu for making sure that the entire farm where the research took place was in order.